Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that function as intracellular effectors of cytokine and growth factor signaling pathways (Darnell, 1997, Science 277(5332):1630-1635). STAT proteins were originally defined in the context of normal cell signaling where STATs have been implicated in control of cell proliferation, differentiation, and apoptosis (Bromberg and Darnell, 2000, Oncogene, 19:2468-2473; Damell et al., 1994, Science 264:1415-1421).
Stat3β is a truncated form of Stat3 that contains the dimerization and DNA binding domain but lacks the transactivation domain (Catlett-Falcone et al., 1999, Immunity, 10:105-115). As a consequence, Stat3β can bind DNA but cannot transactivate gene expression, thus blocking Stat3 signaling in a trans-dominant negative fashion in most cases. Blocking Stat3 by Stat3β in U266 cells, which are cells that are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-x L, down-regulated expression of the Stat3-regulated BCl-XL gene, resulting in a dramatic sensitization of cells to Fas-mediated apoptosis in vitro (Catlett-Falcone et al., 1999, supra).
Recent studies in genetically-deficient mice demonstrate that multiple components of both the innate and adaptive immune system can act as extrinsic tumor suppressors (Kaplan et al., 1998, Immunology 95:7556-7561; Shankaran et al., 2001, Nature 410: 1107). Indeed, tissue disruption, such as that associated with invasion and metastatic spread of cancer, can stimulate pro-inflammatory signals similar to pathogen infection, which activate antigen presenting cells, leading to antigen-specific immune responses. However, the immune system is generally tolerant to established cancers (Fuchs and Matzinger, 1996, Semin. in Immunol. 8:271-280; Pardoll, 1998, Nat Med 4:525-531), suggesting that cancers can develop mechanisms to inhibit production of and/or sensing of immunologic danger signals.
Stat3 is a negative regulator of inflammatory responses, as mice devoid of the Stat3 gene in macrophages and neutrophils produce elevated levels of pro-inflammatory cytokines upon lipopolysaccharide (LPS). induced stimulation of the immune system, leading to development of chronic enterocolitis (Takeda et al, 1999, Immunity 10:39-49). Stat3 is a common point of convergence for oncogenic tyrosine kinases, and constitutively-activated Stat3 enhances tumor cell proliferation and prevents apoptosis (Catlett-Falcone, 1999, Immunity 10:105-115; Grandis et al., 2000, Proc Natl Acad Sci 97:4227-4232; Bromberg et al., 1999, Cell 98:295-303; Bowman et al., 2001, Proc Natl Acad. Sci 98:7319-7324).
2.1 The Immune Response
Cells of the immune system arise from pluripotent stem cells through two main lines of differentiation, the lymphoid lineage and the myeloid lineage. The lymphoid lineage produces lymphocytes, such as T cells, B cells, and natural killer cells, while the myeloid lineage produces monocytes, macrophages, and neutrophils and other accessory cells, such as dendritic cells, platelets, and mast cells. Lymphocytes circulate and search for invading foreign pathogens and antigens that tend to become trapped in secondary lymphoid organs, such as the spleen and the lymph nodes, where such antigens are taken up by antigen-presenting cells (APCs). The interaction between T cells and APCs triggers several effector pathways, including activation of cytotoxic T lymphocytes (CTLs) and stimulation of T cell production of cytokines. CTLs then kill target cells that carry the same class I MHC molecule and the same antigen that originally induced their activation.
2.2 Antigen Presentation
Major histocompatibility complex (MHC) molecules present antigens on the cell surface of antigen-presenting cells. Cytotoxic T lymphocytes then recognize MHC molecules and their associated peptides and kill the target cell. Antigens are processed by two distinct routes depending upon whether their origin is intracellular or extracellular. Intracellular or endogenous protein antigens, i.e., antigens synthesized within the antigen-presenting cell, are presented by class I MHC molecules to CD8+ cytotoxic T lymphocytes. CD8+ CTLs are antigen-specific effector cells derived from pluripotent stem cells via the lymphoid lineage that are important in resisting pathogens, cancer and allograft rejection, and are expressed in most cell types (Terstappen et al., 1992, Blood 79:666-677). On the other hand, extracellular antigenic determinants are presented on the cell surface of “specialized” or “professional” APCs (macrophages, for example) by class II MHC molecules to CD4+ “helper” T cells (see generally, W. E. Paul, ed., Fundamental Immunology. New York: Raven Press, 1984).
Class I and class II MHC molecules are the most polymorphic proteins known. A further degree of heterogeneity of MHC molecules is generated by the combination of class I and class II MHC molecules, known as the MHC haplotype. In humans, HLA-A, HLA-B and HLA-C, three distinct genetic loci located on a single chromosome, encode class I molecules. Because T cell receptors specifically bind complexes comprising antigenic peptides and the polymorphic portion of MHC molecules, T cells respond poorly when an MHC molecule of a different genetic type is encountered. This specificity results in the phenomenon of MHC-restricted T cell recognition and T cell cytotoxicity.
The process of presenting an antigen to T cells involves antigen capture by an APC, either by binding to a receptor or by uptake in the fluid phase. This is followed by proteolytic degradation of the antigen, and formation of a complex between the antigenic peptide and an MHC molecule within the APC (Lanzavecchia, 1996, Curr. Opin. Immunol. 8:348-354). In pathogen-infected cells, proteins of the pathogen are degraded inside the cells, and some of the resulting peptides are transported into the lumen of the endoplasmic reticulum where they form complexes with class I MHC molecules. Additionally, antigens can be chaperoned by heat shock proteins into an endogenous pathway whereby antigenic peptides become associated with class I MHC molecules (Suto et al., 1995, Science 269:1585-1588; Srivastava et al., 1994, Immunogenetics 39:93-98). These class I MHC protein—peptide complexes are then transported to and accumulate on the cell surfaces, where they are recognized by receptors on T cells (Yewdell et al., 1992, Adv. Immunol. 52:1-123; Bevan, 1995, J. Exp. Med. 182:639-641).
Cytotoxic T lymphocytes and helper T cells develop and undergo selection in the thymus. These cells are distinguished by the presence of one of two surface markers, CD4 (helper T cells) or CD8 (CTLs). These lymphocytes circulate in the periphery and become “primed” in the lymphoid organs on encountering the appropriate signals defined by the two signal model originally proposed for B cells (Bretscher & Cohn, 1970, Science 169:1042-1049). The first signal is received through the T cell receptor after it engages antigenic peptides complexed with class I MHC molecules on the surface of APCs. The second signal is provided either by a secreted chemical signal or cytokine, such as interleukin-1 (IL-1), or by a plasma-membrane-bound costimulatory molecule, such as B7. Cytokines, such as interferon-γ, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), and interleukin-12 (IL-12), produced by CD4+ helper T cells, are required.
Helper T cells receiving both signals are activated to proliferate and to secrete a variety of interleukins. CTLs receiving both signals are activated to kill target antigenic cells. However, T cells receiving the first signal in the absence of costimulation become anergized, leading to tolerance (Lamb et al., 1983, J. Exp. Med. 157:1434-1447; Mueller et al., 1989, Annu. Rev. Immunol. 7:445-480; Schwartz, 1992, Cell 71:1065-1068; Mueller and Jenkins, 1995, Curr. Opin. Immunol. 7:375-381).
Cell surface receptor molecules, such as B7, present on the antigen-presenting cell embrane, are recognized by a co-receptor on the cell surface of helper T cells, called CD28, a member of the Ig superfamily. In addition to antigen-specific interactions during antigen presentation, antigen non-specific adhesive mechanisms also operate. These stabilize the binding of T lymphocytes to APC. Receptor molecules on APC, such as ICAM-1/CD54, LFA-3/CD58, and B7, bind corresponding co-receptors on T cells.
2.3 Adoptive Immunotherapy
The cytotoxic T cell response is the most important host response for the control of growth of antigenic tumor cells (Anichimi et al., 1987, Immunol. Today 8:385-389). Studies with experimental animal tumors as well as spontaneous human tumors have demonstrated that many tumors express antigens that can induce an immune response. Some antigens are unique to the tumor, and some are found on both tumor and normal cells. Several factors influence the immunogenicity of the tumor, including, for example, the specific type of carcinogen involved, and immunocompetence of the host and the latency period (Old et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; Bartlett, 1972, J. Natl. Cancer. Inst. 49:493-504). It has been demonstrated that T cell-mediated immunity is of critical importance for rejection of virally and chemically induced tumors (Klein et al., 1960, Cancer Res. 20:1561-1572; Tevethia et al., 1974, J. Immunol. 13:1417-1423).
Adoptive immunotherapy for tumors refers to the therapeutic approach wherein immune cells with antitumor reactivity are administered to a tumor-bearing host, with the objective that the cells cause the regression of an established tumor, either directly or indirectly. Immunization of hosts bearing established tumors with tumor cells or tumor antigens has generally been ineffective since the tumor may have already elicited an immunosuppressive response (Greenberg, 1987, Chapter 14, in Basic and Clinical Immunology, 6th ed., ed. by Stites, Stobo and Wells, Appleton and Lange, pp. 186-196). Thus, prior to immunotherapy, it had been necessary to reduce the tumor mass and deplete all the T cells in the tumor-bearing host (Greenberg et al., 1983, page 301-335, in Basic and Clinical Tumor Immunology, ed. Herbermann R R, Martinus Nijhoff).
Animal models have been developed in which hosts bearing advanced tumors can be treated by the transfer of tumor-specific syngeneic T cells (Mulé et al., 1984, Science 225:1487-1489). Investigators at the National Cancer Institute (NCI) have used autologous reinfusion of peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL), T cell cultures from biopsies of subcutaneous lymph nodules, to treat several human cancers (Rosenberg, S. A., U.S. Pat. No. 4,690,914, issued Sep. 1, 1987; Rosenberg et al., 1988, N. Engl. J. Med., 319:1676-1680). For example, TIL expanded in vitro in the presence of IL-2 have been adoptively transferred to cancer patients, resulting in tumor regression in select patients with metastatic melanoma. Melanoma TIL grown in IL-2 have been identified as CD3+ activated T lymphocytes, which are predominantly CD8+ cells with unique in vitro anti-tumor properties. Many long-term melanoma TIL cultures lyse autologous tumors in a specific class I MHC- and T cell antigen receptor-dependent manner (Topalian et al., 1989, J. Immunol. 142:3714).
Application of these methods for treatment of human cancers would entail isolating a specific set of tumor-reactive lymphocytes present in a patient, expanding these cells to large numbers in vitro, and then putting these cells back into the host by multiple infusions. Since T cells expanded in the presence of IL-2 are dependent upon IL-2 for survival, infusion of IL-2 after cell transfer prolongs the survival and augments the therapeutic efficacy of cultured T cells (Rosenberg et al., 1987, N. Engl. J. Med. 316:889-897). However, the toxicity of the high-dose IL-2 and activated lymphocyte treatment has been considerable, including high fevers, hypotension, damage to the endothelial wall due to capillary leak syndrome, and various adverse cardiac events such as arrhythmias and myocardial infarction (Rosenberg et al., 1988, N. Engl. J. Med. 319:1676-1680). Furthermore, the demanding technical expertise required to generate TILs, the quantity of material needed, and the severe adverse side effects limit the use of these techniques to specialized treatment centers.
Antigen-specific CTL can be primed in vivo by immunization of animals with antigen-expressing cells, or with the antigen plus selected adjuvants (Udono et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:3077-3081; Hadden, 1994, Int. J. Immunopharmacol. 16:703-710).
CTLs specific for class I MHC—peptide complexes could be used in treatment of cancer and viral infections, and ways have been sought to generate them in vitro without the requirement for priming in vivo. These include the use of dendritic cells pulsed with appropriate antigens (Inaba et al., 1987, J. Exp. Med. 166:182-194; Macatonia et al., 1989, J. Exp. Med. 169:1255-1264; De Bruijn et al., 1992, Eur. J. Immunol. 22:3013-3020). RMA-S cells (mutant cells expressing high numbers of ‘empty’ cell surface class I MHC molecules) loaded with peptide (De Bruijn et al., 1991, Eur. J. Immunol. 21:2963-2970; De Bruijn et al., 1992, supra; Houbiers et al., 1993, Eur. J. Immunol. 26:2072-2077) and macrophage phagocytosed-peptide loaded beads (De Bruijn et al., 1995, Eur. J. Immunol. 25, 1274-1285). Fusion of B cells or dendritic cells with tumor cells has been previously demonstrated to elicit anti-tumor immune responses but not T cell priming in vitro (Guo et al., 1994, Science, 263:518-520; Gong et al., 1997, Nat. Med. 3:558-561; Celluzzi, 1998, J. Immunol. 160:3081-3085).
Although tumor progression involves processes such as tissue invasion that can activate inflammatory responses, the immune system largely ignores or tolerates disseminated cancers. This implies that successful tumors must develop specific mechanisms to evade immune surveillance. While much effort has been focused on how tumors resist killing by effector T cells, little is known about mechanisms that block initiation of immune responses during transformation and malignant progression.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.